This project brings the work and expertise of this laboratory on the molecular and cellular pathophysiology of sickle cell disease into collaboration with four other laboratories, each expert in different areas, with the common goal of a detailed description of the structure, interactions and kinetics of assembly of the deoxy-HB S polymers, fibers and gel and the behavior of Hb S in the red cell. We emphasize these objectives: 1) To characterize the interaction sites of the deoxy-Hb S polymers, fibers and gels, with particular attention to important sites susceptible to specific modification, by semisynthetic approaches and direct chemical modification; to the specific cis or trans role of specific residues at different structural levels of the polymer; and how non-S Hbs participate in polymerization; 2) to correlate our micro excluded volume assay of the polymer solubility, the dextran-Csat with Ferrone?s new micro-kinetic assay of polymerization, to permit thermodynamic analyses with this method; 3) to examine further the role of the polymer water compartment (PWC) on the redistribution of 2,3- DPG, ATP, RBC enzymes and small molecules which may affect both the polymerization process and its effects on cell metabolism; 4) to test for conformational changes in deoxy-Hb S polymerized in different conditions, to explain the observed exclusion of DPG, and solubility-lowering effect if IHP 4)to investigate the mechanisms of intracellular instability of Hb S, which generates hemichromes and oxidative membrane damage 5) to assess the role of normal or abnormal (SS-modified) components of the RBC membrane on the kinetics and assembly of intracellular polymer. Each recombinant, modified, and/or cross-linked hybrid tetramer Hb designed specifically to test the roles of individual sites in different levels of polymer and gel structure (Manning, Acharya), will be examined by our micro-assay of equilibrium solubility (dextran-Csat) and correlated with the micro-kinetic assay (Ferrone). Selected species will be examined by Joseph's electron microscopic methods, and by Briehl's methods of direct visualization of fiber growth and assembly. We share the goal of a detailed characterization of the structure and assembly mechanisms of the deoxy-Hb S polymer and gel, in solution and red cells, with the clinical aim of facilitating the design of specific therapeutic agents for sickle cell disease, and the basic aim of advancing a full model description of the mechanism and osmotic effects of protein polymerization and depolymerization in biological systems.